Recombinant adeno-associated virus (rAAV) vectors are limited by the requirement for host-cell DNA synthesis to generate a complementary strand from the single-stranded virion DNA template. This occurs with varying efficiency in different cell types and can be induced through treatment with co-infecting adenovirus, UV irradiation, or other means of DNA damage or cell stress. We have circumvented this limitation using the tendency of AAV to package dimeric inverted repeat DNA molecules when the genome is half the wild-type genome length. These genomes can re-anneal to form ds-DNA upon release from the capsid with no host-cell DNA synthesis required. The self-complementary rAAV (scAAV) transduces more efficiently than the homologous single-strand rAAV and is unaffected by inhibitors of DNA synthesis. In mouse muscle and liver, scAAV transgene expression began sooner, reached higher levels, and was observable at lower doses than conventional single-strand rAAV vectors. Transduction in mouse liver with rAAV had previously been limited to less than 5% of hepatocytes. Using the scAAV, we can transduce approximately 50% of mouse hepatocytes after a single tail-vein injection using the same dose. This illustrates both the quantitative and qualitative advantage of the scAAV vectors. In brain, greater expression levels and saturation of the injected area were achieved when the need for DNA synthesis was eliminated. The scAAV vectors have sufficient genetic capacity for gene therapy applications including delivery of small protein coding genes and ribozyme and anti-sense RNA strategies. Apart from its utility as a vector, scAAV represents a unique intermediate in the rAAV transduction pathway, which can further our understanding of barriers to rAAV transduction. In Aim 1, a liver ischemia-reperfusion model will be used to ask whether the rapid onset of scAAV gene expression will allow its use to deliver an anti-oxidant gene for protection of transplanted tissue and organs. In Aim 2, will focus on whether scAAV vectors can express therapeutic genes in the central nervous system more effectively than single-strand rAAV vectors. The transducing efficiency of scAAV, packaged into different AAV serotypes, will be tested in mouse brain and the feasibility of adapting Tet regulation to scAAV vectors will be tested by separately packaging the transgene and regulatory gene and co-delivering them in a single-injection. This system will be adapted to the delivery of the gene coding iduronate sulfatase for correction of MPS II in a Hunters disease mouse model. In Aim 3, the feasibility of expanding the scAAV coding capacity by using the intrinsic AAV terminal repeat (TR) promoter activity will be tested. The production and purification of scAAV vectors has been streamlined by creating constructs with mutations in one TR, forcing the replication of dimeric genomes. These will be tested for the effects of the mutations on transcription initiation from the terminal repeats.